High frequency of KRAS and EGFR mutation profiles in BRAF-negative thyroid carcinomas in Indonesia

Objective Thyroid cancer incidence has steadily increased in Indonesia. However, data on Kirsten rat sarcoma virus (KRAS) and EGFR mutations in thyroid cancer in Indonesia remain unavailable, except for BRAF-V600E, the most common BRAF gene mutation. This study aimed to analyze KRAS and EGFR mutation profiles in BRAF-V600E negative thyroid cancer samples. Results BRAF-V600E mutations were found in papillary thyroid carcinomas in 40.3% patients with mean age of 53 years old. In BRAF-V600E-negative samples, 41.3% had KRAS mutations with mean age of 55.5 years old. KRAS mutation was found in 52.6% of follicular carcinomas and 47.4% of papillary thyroid carcinomas. Additionally, 45.7% had EGFR mutations in patients with mean age of 50.5 years old. EGFR mutation was found in 71.4% of papillary thyroid carcinoma and 28.6% of follicular carcinoma. Nearly half of the BRAF-V600E negative thyroid carcinoma samples harbored either KRAS or EGFR mutations. This finding suggests that in BRAF-V600E negative thyroid carcinoma samples, testing for RAS and EGFR mutation may be warranted for further therapeutic consideration. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-022-06260-4.


Introduction
Thyroid cancer is the most common endocrine cancer in the world, with Indonesia ranked 9th position in 2010 [1,2]. The currently available prognostic factors in thyroid cancer are the TNM classification of malignant tumors (TNM) staging, age, gender, and histopathology profile (including capsular invasion and angioinvasion) [3]. Although clinical assessment of these factors are known, the frequency and behavior of thyroid cancer vary greatly, ranging from frequent and generally asymptomatic to larger and more advanced papillary tumors.
Mutation in B-Raf proto-oncogene (BRAF) is the gene most often disrupted in thyroid cancer [4]. BRAF-V600E mutation increases disease progression risk in thyroid cancer and correlates with poor prognosis [5,6]. Following BRAF mutations, Ras proteins are related to follicular-patterned neoplasms and are the second most common genetic alteration seen in thyroid carcinomas [7]. The RAS proteins are encoded by three human RAS genes: Kirsten rat sarcoma viral (KRAS), neuroblastoma (NRAS) and Harvey (HRAS). However, despite extensive research, their profile remains largely uncharacterized especially in Indonesia [8]. Epidermal growth factor receptor (EGFR), on the other hand, have been implicated in various cancer pathogenesis, including thyroid cancer [9,10]. EGFR was also implied to be important for thyroid carcinoma proliferation and metastasis, and high EGFR level was reported in malignant thyroid cancer with wild type BRAF [10][11][12]. This suggest that EGFR mutation profile in thyroid cancer should not be overlooked. Overall, investigation for different mutation profiles on thyroid carcinomas is still required to understand the key mutation that occurred.
With high mutation prevalence in thyroid cancer, a preoperative approach is required to assist clinicians in determining the extent to which surgical intervention and adjuvant therapy are required. However, research focusing on mutation profile, including the presence of KRAS and EGFR mutations in addition to BRAF mutations in thyroid cancer, is still rarely described, particularly in Indonesia. This study is expected to be a reference in making diagnostic and treatment decisions in thyroid cancer.

Study design
This study was an retrospective observational study design. Samples were obtained from the archive between 2013 and 2019 and used to investigate BRAF, KRAS, and EGFR mutations in thyroid carcinoma samples. Paraffin blocks from patients histopathologically diagnosed with primary thyroid carcinoma (papillary thyroid carcinoma of all variants, and follicular thyroid carcinoma) were included. Samples were obtained from Department of Anatomical Pathology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, and CITO Pathology Laboratory. Histological evaluation was reviewed by two pathologists and noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) samples were excluded. The paraffin blocks were required to contain more than 50% area of tumor. Exclusion criteria were damaged paraffin blocks and paraffin blocks stored at temperatures other than room temperature. This study initially collected 87 primary thyroid carcinoma cases. Of those, highly degraded DNA samples were excluded, leaving only 77 paraffin blocks meeting the criteria. The samples were then examined for BRAF, KRAS

DNA extraction
DNA extraction was performed from paraffin blocks using tissue genomic DNA mini kit (GeneAll ® Clinic SV mini Cat. 108-101, Seoul, Korea) according to the manufacturer's instructions.

Real-time polymerase chain reaction (qRT-PCR)
DNA samples were assayed using AmoyDx BRAF-V600 mutation detection kit (Amoy Diagnostics, Xiamen, China) [13]; AmoyDx human EGFR gene mutations fluorescence PCR diagnostic kit (Amoy Diagnostics, Xiamen, China), detecting T970M, L858R, L861Q, S7681, G719S, G719A, G719C, 3 insertions in exon 20, and 19 deletions in exon 19 [14]; and AmoyDx KRAS Mutations Detection Kit (Amoy Diagnostics, Xiamen, China) [15]. Quantitative PCR was performed using Bioneer Exicycler ™ 96 Real-Time Quantitative Thermal Block. The assays and PCR condition were as manufacturer's recommendation. Briefly, region of interest within the DNA sequence is amplified using the polymerase chain reaction, in which the DNA of the mutant gene is amplified by specific primers and detected by novel probes. The amplified region of interest is heated gradually until the double stranded DNA breaks. The cycle threshold (Ct) values at which the signal is detected above the fluorescence background of the respective mutation testing kit were used to determine whether a sample is positive or negative. Internal control reagents are intended to detect the presence of inhibitors, which can result in false-negative results. The real-time PCR was performed with 1 cycle denaturation at 95 °C for 5 min, 15 cycles annealing at 95 °C for 25 s, 64 °C for 20 s, and 72 °C for 20 s, and 31 cycles extension at 93 °C for 25 s, 60 °C for 35 s, and 72 °C for 20 s.

Data analysis
All data presented were made using Microsoft Excel 2020 for Mac OSX. Categorical data were expressed as frequency and percentage, and quantitative data were expressed as mean.

Clinicopathologic characteristics of the thyroid carcinoma samples
Characteristics of the samples are shown in Table 1. A total of 37 (48.1%) samples were from males and 40 (51.9%) samples were from females. The mean age of the patients was 53.6 ± 13.4 years old. The histopathological types were 57 samples of papillary type (74%) and 20 samples of follicular type (26%). The papillary type was consisted of 32 classic variant (56.1%), 19 follicular variant (33.3%), and three samples of tall cell and columnar cells variant (5.3% and 5.3%, respectively).

BRAF-V600E mutation status in thyroid carcinoma samples
A total of 31 (40.3%) samples showed BRAF positive mutation consisted of 5 males and 26 females ( Table 2). The mean age of the patients was 53.0 ± 14.7 years old. Histopathological types of BRAF positive mutation samples were of papillary type, with classic variant of 26 samples (76.5%) and follicular of 5 (14.7%) samples.

KRAS and EGFR mutation were detected in BRAF-V600E negative samples
Further examination of the BRAF-negative samples revealed that 19 (41.3%) were KRAS positive, with 15 of males and 4 of females ( Table 3). The mean age was 55.5 ± 12.4 years old. Histopathological types of KRAS positive mutation samples were of papillary type 9 (47.4%) samples and follicular type 10 (52.6%) samples. The papillary type consisted of two (10.5%) classic variant samples, three (15.8%) follicular samples, three (15.8%) tall cell samples, and one (5.2%) columnar cell variant. Capsular invasion was observed in 84.2% of KRAS positive samples, while vascular invasion was observed in 78.9% of samples.
BRAF negative samples also showed EGFR positive mutation in 21 (45.7%) samples consisted of 13 males and 8 female samples. The mean age was 50.5 ± 12.5 years old. Histopathological types of EGFR positive samples were 15 papillary type (71.4%), and six follicular type (28.6%). The papillary type consisted of four classic variant samples (19%), nine follicular (42.9%) samples, and two (9.5%) columnar cell variants. Capsular invasion was found in 81.0% of EGFR positive samples, with vascular invasion in 57.1% of the samples. Notably we also identified one papillary thyroid carcinoma of follicular variant with both KRAS and EGFR mutation (Additional file 1: Table S2).

Discussion
The main findings of this study were (1) The thyroid cancers samples were consisted of papillary type carcinoma (74.0%) and follicular type carcinoma (26.0%), (2) BRAF-V600E mutation were found in 40.3% of thyroid carcinoma samples, (3) KRAS mutation was found in 41.3% of BRAF negative thyroid carcinoma samples, with predominant follicular type, and (4) EGFR mutation was observed in 45.7% of BRAF negative thyroid carcinoma samples, with predominant papillary type.
We observed that thyroid cancers samples were mainly of papillary type. Papillary thyroid carcinoma was the most common carcinoma arising from the thyroid gland [16]. In South Korea, for example, papillary thyroid carcinoma accounted for the greatest age-standardized incidence rate [16]. Even though it is considered as malignancy with good prognosis, recent study with 15-year median follow up showed significantly lower survival percentage in papillary thyroid carcinoma with BRAF mutation [17]. Therefore, determining the thyroid carcinoma mutation profile is important in determining patient prognosis.
We observed that BRAF mutation was found in 40.3% of all thyroid samples, which is comparable to previous   [20]. The number of deaths came at third position following China and India. BRAF mutations were associated with thyroid cancer pathogenesis, leading to unregulated cell growth and carcinogenesis as well as extrathyroidal extension and advanced stage [18,21]. This implies that identifying mutations in thyroid cancer is critical for better diagnosis and, potentially, better treatment. The RAS proteins transmit extracellular signals that promote cell proliferation, differentiation, and survival [22,23]. The EGFR signaling plays a role in signaling the downstream BRAF. Therefore, we conduct testing on KRAS and EGFR mutation in thyroid cancer patients who did not harbor BRAF mutation. We observed that KRAS mutation occurred in 41.3% of the BRAF negative thyroid cancer samples, and 52.6% of it was found in follicular thyroid carcinoma. Similar finding was reported in Japan in which 57% of follicular carcinomas harbored RAS mutations [8]. Another study in Saudi Arabia assessing proliferative thyroid lesions reported that 32% of mutations affected the KRAS codon 61 [24]. Consistently, we reported high KRAS prevalence and this may be higher since we reported KRAS mutations only in BRAFnegative thyroid cancer samples. Point mutations in the RAS or BRAF proto-oncogenes were detected in almost 70% of papillary thyroid carcinomas in nearly mutually exclusive manner [25]. This further shows that BRAFnegative samples are prone to have other mutations, including RAS mutations. In addition, this could be the factor contributing to poor prognosis in follicular thyroid carcinoma, as RAS mutation is linked to significant lower outcome [26]. Therefore, establishing the necessary mutation testing steps in thyroid carcinoma is important to determine the need for early intervention.
We also observed that 45.7% of BRAF negative thyroid carcinoma samples had EGFR mutation, with 71.4% was found in papillary thyroid carcinoma. This finding is similar to a study in United States with most of EGFR mutation was found in papillary thyroid carcinoma (46.2%), followed by follicular type (29.6%) [27]. EGFR is important for extracellular signal transduction from the surface into cells to mediate cell proliferation and apoptosis [27]. Its overexpression is common in breast, lung, and bladder cancer. Fisher et al. found that EGFR mutations correlates with advanced stage and lymph node metastases which may contribute to papillary thyroid carcinoma aggressiveness [27]. These suggest that EGFR mutation profile in thyroid carcinoma should not be excluded.
Interestingly, coexistence mutations (both KRAS and EGFR mutations) were found in 1 of our BRAF negative thyroid carcinoma sample. In non-small cell lung cancer (NSCLC), for example, coexistence mutations of KRAS and EGFR are relatively rare [28]. This phenomenon was suggested to occur due to heterogeneity in malignant cells where some subpopulations of the cells had different mutations. The EGFR coexistence with KRAS mutation resulted in significantly shorter progression free survival [29]. Although KRAS and EGFR mutations have been well investigated in NSCLC, however, study of these mutations on thyroid carcinoma is still limited. These findings emphasize the importance of identifying KRAS and EGFR mutations following the most common genetic alteration in thyroid cancer, BRAF.
Our study highlights the frequent BRAF, KRAS, and EGFR mutations in thyroid carcinomas. Of those BRAF-V600E negative samples, almost half had either KRAS or EGFR mutations. Although differentiated thyroid cancers have favourable prognosis, about 30% will experience recurrent disease after 10 years [30]. BRAF, KRAS, and EGFR mutations in recurrent thyroid cancer may benefit from targeted therapies and further study on these mutations with their risk of recurrence, distant metastasis, and overall prognosis in thyroid cancers may be required.

Conclusion
We discovered that either KRAS or EGFR mutations can be found in nearly half of the BRAF negative thyroid carcinoma samples. Thyroid carcinoma-related death is still a burden in Indonesia. These findings suggest that additional mutational pathways, such as KRAS and EGFR, should be tested in BRAF-V600E negative thyroid carcinoma samples. The breakthrough in molecular testing guidelines might provide help in determining the prognosis of thyroid cancer and might aid clinicians in determining early intervention for better outcome.

Limitation
Due to sample availability, this study only comprised a small number of specimens. In addition, the data obtained in this study were pathology data, whereas with clinical and survival data, a better association can be concluded. Future research should have a large enough sample size to establish enough statistical power for analysis.